The route of cellular entry for most conventional drugs is diffusion across the biological membrane. For this reason, drugs tend to be small (MW<500) and amphipathic, containing both hydrophobic and hydrophilic functionalities. These characteristics engender molecules with water solubility, while allowing them to cross the nonpolar lipid bilayer of the cell membrane. However, many potential drugs, including those used in gene therapy are too hydrophilic and/or too large to be delivered to cells by diffusion across a cell membrane. For this reason, a major barrier to gene therapy is the delivery of the large hydrophilic drugs to the cellular cytoplasm or nucleus.
The route of entry into cells for most membrane impermeable molecules is endocytosis. After internalization, the contents of an endosome are typically recycled back to the cell surface or delivered to another intracellular membrane bound vesicle, such as a lysosome. Delivery to a lysosome occurs concomitantly with a drop in pH of the vesicle interior, from pH about 7.5 outside the cell, to pH 7-6 in early and late endosomes, to pH about 5 or less in lysosomes. To deliver endocytosed membrane impermeable molecules to the cell cytoplasm, the molecule must therefore be co-delivered with compounds that facilitate release of the molecule from an internal membrane bound vesicle or facilitate membrane permeability of the molecule.
Release of endosomal contents can occur through disruption of the vesicle membrane or rupture of the vesicle. Agents used to accomplish endosomal release include compounds which are proposed to act as proton sponges and membrane active compounds that directly disrupt membrane structure. These compounds, e.g., adenoviral coat proteins, often rely upon the environment of the endosome/lysosome to trigger their activation. For example, these compounds may be substrates for lysosomal degradative enzymes such as proteases, nucleases and glycosylases. Proteolysis can result a activation of a membrane active compound which then destabilizes the bilayer.
The drop in pH as an endosome matures into a lysosome may also be utilized to trigger membrane disruption and content release. pH-sensitive compounds, including polymers and lipids, have found broad application in the area of drug delivery.
Agents that are weakly basic, pKa 5-7, can be reversibly protonated in the acidic environment of the endosome. Examples include chloroquine, polyethyleneimine, and histidylated poly-L-lysine. The effect of these buffering compounds is to increase the number of protons required for a drop in pH. It is postulated that the increased number of protons, and as a consequence their counterions, causes an increase in the osmotic pressure of the endosome that leads to membrane rupture, the proton sponge effect.
Another mechanism for pH-dependent membrane disruption is the use of agents whose interaction with a membrane is dependent upon protonation, e.g. cholesterol hemisuccinate, viral coat peptides and their derivatives, and polypropylacrylic acid (PPA). A common characteristic of these agents is that they are carboxylic acid- and hydrophobic group-containing molecules that become less charged as the pH drops. The decrease in charge renders the molecules more hydrophobic, and thus more membrane disruptive.
Still other compounds rely on pH dependent cleavage events to facilitate membrane disruptive activation, prodrug activation, or drug release. pH-sensitive polymers have found broad application in the area of drug delivery, exploiting various physiological and intracellular pH gradients for the purpose of controlled or targeted release of drugs (both low molecular weight conventional drugs as well as membrane impermeable biologically active compounds). The controlled release of pharmaceuticals after their administration is under intensive development. pH sensitivity can be broadly defined as any change in polymer's physico-chemical properties over certain range of pH. A more narrow definition demands significant changes in a compound's or polymer's interaction with biological components or its ability to retain (release) a bioactive substance (drug) in a physiologically tolerated pH range (usually pH 5.5-8).
Drugs may be administered to a patient in an inactive form, a called a prodrug. The prodrug is converted into the biologically active compound upon interaction with specific enzymes in the body or upon exposure to specific environments in the body. For example, anticancer drugs are quite toxic and are administered as prodrugs which do not become active until they come in contact with the cancerous cell (Sezaki et. al. 1989). Studies have found that the pH in solid tumors is 0.5 to 1 unit lower than in normal tissue and the use of pH-sensitive polymers for targeting tumors has been shown in vitro (Potineni et al 2003). pH-sensitive polymers have also been used in conjunction with liposomes for the triggered release of an encapsulated drug. For example, hydrophobically-modified N-isopropylacrylamide-methacrylic acid copolymer can render regular egg phosphatidyl chloline liposomes pH-sensitive by pH-dependent interaction of grafted aliphatic chains with lipid bilayer (Meyer et al. 1998).
Polyions can be divided into three categories based on their ability to donate or accept protons in aqueous solutions: polyacids, polybases and polyampholytes. Polybases (polycations) have found broad applications as transfection agents for nucleic acid delivery applications due to the fact they readily interact with polyacids (i.e., nucleic acid). An example is polyethyleneimine (PEI). This polymer facilitates nucleic acid condensation, and electrostatic adsorption on the cell surface followed by endocytosis. Subsequent endosomal release of the nucleic acid is proposed to occur though the so-called proton sponge effect.
Polycations can facilitate DNA condensation. The volume which one DNA molecule occupies in a complex with polycations is lower than the volume of the free DNA molecule. A significant number of multivalent cations with widely different molecular structures have been shown to induce condensation of DNA. Multivalent cations with a charge of three or higher have been shown to condense DNA. Analysis has shown DNA condensation to be favored when 90% or more of the charges along the sugar-phosphate backbone are neutralized. The electrophoretic mobility of nucleic acid-polycation complexes can change from negative to positive in excess of polycation.
The size of a DNA/polymer complex is important for gene delivery in vivo. In terms of intravenous injection, the polynucleotide-containing complex needs to be able to cross the endothelial barrier and reach the parenchymal cells of interest. The largest endothelia fenestrae (holes in the endothelial barrier) occur in the liver and have an average diameter of 100 nm under normal conditions. In other organs, the endothelium can be described as a structure that has a large number of small pores with a radius of 4 nm and a low number of larger pores with a radius of 20-30 nm. The size of the DNA complexes is also important for the cellular uptake process. Since endocytic vesicles typically have an internal diameter of about 100 nm, complexes smaller than about 100 nm in diameter are preferred.
Depending upon conditions used to condense polynucleotide, three main types of structures can be formed: toroidal structures containing as little as a single polynucleotide molecule, microaggregates that remain in suspension and can be toroids, rods or small aggregates, and large aggregates that sediment readily.
A polycation also can form a cross-bridge between an anionic polynucleotide and the anionic surface of a cell. As a result the main mechanism of polynucleotide/polycation complex translocation to the intracellular space may be non-specific adsorptive endocytosis. Polycations are furthermore a convenient linker for attaching functional groups. Polymer/polynucleotide complexes can also protect the polynucleotide against nuclease degradation.
Optimal transfection activity in vitro and in vivo can require an excess of polycation molecules. However, the presence of excess polycations may be toxic to cells and tissues. Moreover, the non-specific binding of cationic particles to all cells interferes with cell type specific targeting. Positive charge also has an adverse influence on biodistribution of the complexes in vivo.
Several modifications of DNA/cation particles have been created to circumvent the nonspecific interactions of the DNA/cation particle and the toxicity of cationic particles. Examples of these modifications include attachment of steric stabilizers. Another example is recharging the DNA particle by the addition of polyanions which interact with the cationic particle, thereby lowering its surface charge, i.e. recharging of the DNA particle (U.S. application Ser. No. 09/328,975). Another example is cross-linking the polymers and thereby caging the complex (U.S. application Ser. Nos. 08/778,657, 09/000,692, 9/724,089, 09/070,299, and 09/464,871).
Linkages that are rapidly cleavable or reversible under specific environments, such as the reduced pH of an intracellular endosome/lysosome or tumor, are useful in developing deliver vectors for a variety of biologically active compounds. The acetal linkage has been used extensively as an acid-labile bond in the delivery of drugs. The acetal bond has been used in the construction of drug carriers and to link drug with carriers. Acetals have also been used to construct acid-cleavable surfactants, to separate the detergent into hydrophobic tail and hydrophilic head group. However, acetal linkages created to date have half-lives of hours to days in aqueous conditions at pH 4-7. Acetals which cleave at faster rates would make better linkages agents in certain applications.